Systemic mastocytosis (SM), a clonal myeloproliferative disease with variable clinical manifestations, is associated in most cases with the D816V mutation in KIT. The identification of the KIT D816V mutation in patients with systemic mastocytosis has gained a major prognostic significance in the last several years, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. Imatinib was shown to be ineffective in patients carrying KIT D816V mutation, but effective in cases displaying some other c-kit mutations. Detection of the KIT D816V mutation in patients with SM generally requires bone marrow testing;however, in some patients the mutation is also detectable in peripheral blood. We analyzed purified hematopoietic cell lineages from the peripheral blood of 21 patients with SM. The number of cell types carrying the mutation significantly correlated with the SM variant. Patients with indolent SM had either no detectable mutation or fewer cell lineages involved as compared to patients with more severe disease. Multilineage involvement significantly correlated with increased serum tryptase levels and the extent of bone marrow mast cell infiltrate. This study indicates that detectable KIT D816V mutation in the peripheral blood, particularly in multiple hematopoietic lineages, is associated with more severe forms of systemic mastocytosis. Cooperating genetic events are likely to contribute to the phenotypic diversity of KIT-D816V SM. Therefore, 44 patients with KIT-D816V SM were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations. Activating NRAS mutations were identified in 25% of patients with aggressive disease but were absent in indolent SM. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity. Most agents in clinical trials for aggressive mastocytosis target KIT and, despite potent in vitro activity, have displayed modest clinical efficacy. Therefore, the finding of activating mutations with the potential to cooperate in disease pathogenesis has significant therapeutic implications. KIT has several variants as the result of alternative mRNA splicing. Two isoforms differ by the presence or absence of four amino acids (GNNK) at the juxta-membrane region of the extracellular domain. Differences in signal transduction and biological activities between these two isoforms are reported. The predominant form, GNNK-, demonstrates stronger and more rapid downstream signaling in vitro as well as tumorigenicity in mice. We analyzed the ratio of GNNK- to GNNK+ transcripts in bone marrow mononuclear cells from 25 patients with systemic mastocytosis and 16 normal controls. A relative increase in the GNNK+ variant was observed in more severe disease and KIT D816V specific transcripts displayed a relative GNNK- predominance. This molecular study has significantly increased our understanding of KIT gene regulation in systemic mastocytosis, specifically in the context of KIT activating mutations. Moreover, this approach may have direct prognostic utility by identifying those patients with more aggressive forms of systemic mastocytosis. We continue to make new clinical observations in SM as well. Recently, we observed that soluble IL-5R alpha was significantly elevated in patients with systemic mastocytosis with or without eosinophilia. Although sIL-5R alpha levels were correlated with serum tryptase levels in these patients, eosinophil activation, as assessed by CD69 expression on eosinophils and serum EDN levels, was increased. Whether mast cells are directly responsible for the increase in sIL-5R alpha in mastocytosis patients and/or act indirectly by stimulating eosinophils remains to be elucidated.